The present invention relates to the field of genetic engineering, especially to the transportation of exogenous materials by means of virus particles.
The gene transfer technology to artificially introduce an exogenous gene into cells is an important technology not only as a fundamental technology to analyze a variety of biological phenomena but also as one which leads to useful applications such as gene therapy and production of beneficial animals. Generally, two methods have been used for gene transfer. One is a biological method using a virus having an exogenous gene, and the other is a physical method in which an exogenous gene is physically introduced into cells.
The method using a virus is based on the principle that a cell is infected with a recombinant virus in which the gene of interest is incorporated, and the entire recombinant virus genome integrates into the genome of the host cell. This method is currently attracting much attention as a technological basis for gene therapy for such diseases as Lesch-Nyhan syndrome and adenosine deaminase (ADA) deficiency. However, it has been pointed out that the method has various problems such as the pathogenicity of the virus since it utilizes the biological properties of the virus itself. For this reason, modified retroviral vectors without the regions associated with the viral pathogenicity and replication have currently being developed. However, these modified vectors have yet many problems that they might still cause some undesirable effects on cells, and they can infect only dividing cells.
Therefore, physical methods to introduce non-viral vectors are now used as well as the above-mentioned methods using viruses. In one of the established physical methods, non-viral vectors are introduced into cells in combination with chemicals such as calcium phosphate, DEAE-dextran, polycations, or liposomes. However, these physical methods have such problems that the transfection efficiency of genes into cells is low, and that the exogenous gene on a non-viral vector thus transfected does not reach the cell nucleus in many cases. Therefore, the methods have many difficulties to be overcome for being applied to gene therapy.
Recently, it was reported that the proteins which are transported into the nucleus of eukaryotic cells and function there have a specific amino acid sequence that functions as a signal (NLS: nuclear localization signal) for their transportation into the nucleus (G. Garcia-Bustos et al., Biochem. Biophys. Acta 1071: 83-101 (1991)). Moreover, it was also reported that attaching the nuclear localization signal to a protein that normally does not translocate to the nucleus will confer the nuclear translocation activity on this protein (R. E. Lanford et al., Cell 46: 575-582 (1986), Y. Yoneda et al., Exp. Cell. Res. 170: 439-452 (1987), D. Chelsky et al., Mol. Cell. Biol. 9: 2487-2492 (1989)). Based on this knowledge, researches have been made using the nuclear localization signal so that the gene introduced by physical methods can reach the nucleus with a high probability. That is, the techniques are studied to condense DNA as close as possible to 40 nm, the size of the nuclear membrane pore, attach the nuclear localization signal to this condensate, and thereby actively transport the DNA to the nucleus. For example, efforts have been made to make DNA more compact by using proteins such as HMG-1 and histones, as well as poly-L-lysines (Jose C. Perales et al., E. J. B. 266: 255-266 (1994)), and cationic liposomes (J. Zabner et al., J. B. C. 270: 18997-19007 (1995)).
However, the synthetic chemical approach had problems with solubility and homogeneity of the complex with DNA, and with the varying degrees of condensation of DNA dependent on the salt concentration. Moreover, construction of the complex is possible only under highly alkaline conditions and impossible under physiological conditions, which has been one of the problems to be solved for practical use.
It has been suggested that, on the viruses that infect animals such as adenovirus and SV40, the nuclear localization signals exist in their capsid proteins, and they function to actively translocate their DNA at the early stage of infection (Urs. F. Greber and Harumi Kasamatsu, Trends in Cell Biology 6: 189-195 (1996)). It has been also suggested that the SV40 particle with its diameter of 45 nm invade the nucleus in the form of virion (K. Hummeler et al., J. Virol. 6: 87-93 (1970)). Furthermore, MS-2 phage is reported to have a transport system in which exogenous substances are enveloped by the capsid (International Application published in Japan No. Hei-508168). However, any transport system using virus particles, which is capable of using long chain DNA and translocating the DNA into the nucleus, has not been reported.
An objective of the present invention is to provide a system that enables delivering genes introduced into cells to the nucleus. More specifically, the objective of the invention is to provide a xcex phage with a nuclear localization signal exposed on the outer surface of its head, and capable of packaging long chain DNA.
In order to translocate long chain DNA into the nucleus, it is necessary to condense the DNA to nearly 40 nm, the size of nuclear membrane pore. The present inventors paid attention to the head of a xcex phage, which is able to compactly package desired long chain DNA in vitro and to protect the DNA from the attack by external DNases, and used it as a carrier of the DNA. Furthermore, we paid attention to the phenomena that the viruses that infect animals can invade the nucleus in the form of virion in virtue of nuclear localization signals in their capsid proteins, and attempted to actively transport DNA into the nucleus by preparing and using the xcex phage head to which a nuclear localization signal has been attached. More specifically, we used the following steps.
First, we constructed a vector that expresses a fusion protein between the gpD protein, which is one of the proteins to constitute the xcex phage head, and the nuclear localization signal sequence, transformed Escherichia coli with this vector, then infected the transformants with a mutant xcex phage incapable of expressing gpD in the E. coli cells (hereinafter designated as xe2x80x9cD amber phagexe2x80x9d). By plaque formation analysis and western blot analysis using an anti-gpD antibody, we have confirmed that the mutant phage was complemented by the fusion protein between the gpD protein and the nuclear localization signal sequence expressed by the vector and, a xcex phage having the nuclear localization signal attached to its head was obtained. That is, we have found that the fusion protein expressed in E. coli has been complementarily integrated into the phage head which does not express the protein.
Next, we have obtained a similar result by introducing the vector that expresses the fusion protein between the gpD protein and the nuclear localization signal sequence into the E. coli lysogenized by the mutant xcex phage, and by heat-inducing the lysogenic phage. More specifically, we introduced the vector that expresses the above fusion protein into the E. coli lysogenized by the D amber phage, and heat-induced the transformants. As the result, the phage whose head has not incorporated the fusion protein and consists of the gpE protein became sensitive to EDTA, while the phage which has incorporated the fusion protein exhibited resistance to EDTA. Next, we treated the resulting phage with EDTA and measured the titer. As a result, it was revealed that the phage packaged with 80% genome size DNA was constructed, and that the fusion protein was incorporated in the phage head. In addition, we have confirmed that the phage had the nuclear localization signal exposed on the outer surface of the head. We also confirmed that the phage incorporating the fusion protein was formed in the same manner even when we used 100% genome size DNA. Furthermore, we introduced the phage having the nuclear localization signal exposed on the outer surface of its head into HEL-R66 cells, which are human fetal lung cells, by microinjection and proved that the phage has a nuclear translocation activity, thereby completing the present invention.
Therefore, the present invention relates to a xcex phage capable of packaging macromolecules such as long chain DNA and having a nuclear translocation activity.
More specifically, it relates to:
(1) a phage or its head having a protein containing a nuclear localization signal as a component of the head,
(2) the phage or its head of (1), wherein said nuclear localization signal comprises any one of the sequences described in SEQ ID NO: 1 to SEQ ID NO: 4.
(3) the phage or its head of (1), wherein said phage is a xcex phage,
(4) the phage or its head of (3), wherein said protein containing the nuclear localization signal is a fusion protein between the nuclear localization signal and a phage head protein,
(5) the phage or its head of (4), wherein said phage head protein is D protein of a xcex phage,
(6) a fusion protein between a nuclear localization signal and a protein that forms a phage head,
(7) the fusion protein of (6), wherein said nuclear localization signal comprises any one of the sequences described in SEQ ID NO: 1 to SEQ ID NO: 4.
(8) the fusion protein of (6), wherein said phage is a xcex phage,
(9) the fusion protein of (6), wherein said phage head protein is the D protein of a xcex phage,
(10) a DNA encoding any one of the proteins of (6) to (9),
(11) a vector containing the DNA of (10),
(12) a bacterial host carrying the vector of (11), (13) the bacterial host of (12), wherein said host is Escherichia coli, 
(14) a kit for transforming cells, wherein said kit comprises the bacterial host of (12) or (13), and (b) a phage from which a head protein contained in a fusion protein expressed in said host has been derived, wherein said phage cannot express said head protein in said bacterial host,
(15) the kit of (14), wherein said phage is a xcex phage,
(16) the kit of (14), wherein said head protein contained in the fusion protein expressed in the bacterial host is D protein of a xcex phage,
(17) a method for translocating a desired substance into the nucleus of a desired cell, wherein said method comprises: (a) packaging into the phage or into its head of (1) the desired substance to be translocated to the nucleus, and (b) introducing said phage or its head into the desired cell,
(18) the method of (17), wherein said desired substance is a nucleic acid,
(19) the method of (17), wherein said phage is a xcex phage,
(20) the method of (17), wherein said cell is a mammalian cell.
The present invention relates to the technology to package an exogenous material into the head of a phage to which a nuclear localization signal is attached, introduce the phage into a desired cell in which the exogenous material is to function, and translocate the exogenous material together with the phage particle into the nucleus of the target cell.
The nuclear localization signal used in the present invention is not particularly limited as far as it has the activity to translocate a substance to which the signal sequence is attached into the nucleus. For example, in the case of translocating the xcex phage particle into the nucleus, it is preferable to use the nuclear localization signal of SV40 VP1, SV40 large T antigen, or hepatitis D virus xcex4 antigen, or a sequence containing xe2x80x9cPKKKRKVxe2x80x9d (SEQ ID NO: 4)(by the single letter representation of amino acids as is found in Encyclopedia of Biochemistry, 2nd ed.) that is the minimum unit having the nuclear translocation activity within the nuclear localization signal of SV40 large T antigen.
The phage used in the present invention is not particularly limited as far as an exogenous material can be packaged into its head. Phages such as a xcex phage and an M13 phage can be used.
A number of methods can be used to prepare the phage whose head is constituted by a protein containing the nuclear localization signal. For example, one can chemically bind the nuclear localization signal sequence to a phage head protein, or combine a DNA encoding the nuclear localization signal sequence with gene encoding a phage head protein and incorporate it into a vector, express it as a fusion protein a bacterial host, and proliferate in the host a mutant phage that cannot express the head protein, thereby constructing the phage head. There are no limitation to the vectors that can be used in the above methods, and various vectors can be used. The bacterial host is not particularly limited as far as the phage used in the method can proliferate in the host. For example, when a xcex phage is used, a variety of E. coli strains in which the phage can proliferate can be used. The nuclear localization signal sequence may chemically bind to the phage head protein, directly or via a cross-linking agent or a spacer peptide. The DNA encoding the nuclear localization signal sequence and the gene encoding a phage head protein may be combined directly or through a spacer nucleotide.
The head protein used in the above methods non-limitedly include gpD protein or gpE protein when the phage is a xcex phage, and gene 3 protein when the phage is M13.
In the present invention, the phage is introduced into the cell after packaging an exogenous material. As for packaging, the method of Ishiura et al. (Gene 82: 281-289 (1989)) or the method of Sternberg et al. (Japanese Patent No. Hei 59-500042) can be used. As for the exogenous material, a gene, a gene fragment, ribozyme, an antisense gene, or any other substance to make it function in the nucleus can be used. For example, when gene therapy is performed, it will be effective to use the normal counterpart of a defective gene. When the function of a specific gene is analyzed, it will be effective to use an antisense gene against the gene. Also, if one wishes to create transgenic animals, it will be effective to introduce a gene which is associated with the phenotype to be conferred into them. It should be noted that the present invention enables packaging a long chain nucleic acid such as a gene with its upstream region.
The method to introduce the phage that has packaged an exogenous material, includes the microinjection method, the lipofection method, the liposome method, the HVJ-liposome method, the immuno-liposome method, the pH-sensitive liposome method, the erythrocyte ghost method, the DEAE-dextran method, the method utilizing endocytosis of a receptor on the cell surface, the method utilizing a specific antigen on the cell surface, the method utilizing a synthetic macromolecular carrier, the method utilizing a particle gun, etc. There is no particular limitation to the cells into which the phage packaging an exogenous material is introduced, and various cells can be used depending on the purposes.